The mutagenic and carcinogenic effects of electrophiles such as N-methyl-N-nitrosourea are mainly due to the O6-alkylation of guanine in DNA. To protect themselves against DNA-alkylation, mammals and bacteria possess a protein, O6-alkylguanine-DNA alkyltransferase (AGT) which repairs these lesions. AGT transfers the alkyl group from the position O-6 of alkylated guanine and guanine derivatives to the mercapto group of one of its own cysteines, resulting in an irreversibly alkylated AGT. The underlying mechanism is a nucleophilic reaction of the SN2 type which explains why not only methyl groups, but also benzylic groups are easily transferred. As overexpression of human AGT (hAGT, SEQ ID NO:1) in tumour cells is the main reason for resistance to alkylating drugs such as procarbazine, dacarbazine, temozolomide and bis-2-chloroethyl-N-nitrosourea, inhibitors of AGT have been proposed for use as sensitisers in chemotherapy (Pegg et al., Prog Nucleic Acid Res Mol Biol 51:167-223, 1995). U.S. Pat. No. 5,691,307 describes O6-benzylguanines carrying various substituents in the benzyl group, and their use for depleting AGT levels in tumor cells and thereby increasing responsiveness to alkylating anti-tumor drugs. Likewise, WO 97/20843 discloses further AGT depleting compounds representing O6-benzyl- and O6-heteroarylmethyl-pyrimidine derivatives.
DE 199 03 895 discloses an assay for measuring levels of AGT which relies on the reaction between biotinylated O6-alkylguanine derivatives and human AGT which leads to biotinylation of the AGT. This in turn allows the separation of the AGT on a streptavidin coated plate and its detection, e.g. in an ELISA assay. The assay is suggested for monitoring the level of AGT in tumour tissue and for use in screening for AGT inhibitors.
Damoiseaux et al., ChemBiochem 4:285-287, 2001, disclose modified O6-alkylated guanine derivatives incorporated into oligodeoxyribonucleotides for use as chemical probes for labelling human AGT, again to facilitate detecting the levels of this enzyme in cancer cells to aid in research and in chemotherapy.
WO 02/083937 discloses a method for detecting and/or manipulating a protein of interest wherein the protein is fused to AGT and the AGT fusion protein contacted with an AGT substrate carrying a label, and the AGT fusion protein detected and optionally further manipulated using the label. Several AGT fusion proteins to be used, general structural principles of the AGT substrate and a broad variety of labels and methods to detect the label useful in the method are described. Although other forms of AGT are mentioned, only human AGT is exemplified.
PCT/EP03/10859 (WO 2004/031404) describes particular AGT fusion proteins to be used in the mentioned method for detecting and/or manipulating a protein of interest, labelled fusion proteins obtainable by this method, and the method using the particular AGT fusion proteins.
PCT/EP03/10889 (WO 2004/031405) discloses additional AGT substrates carrying a label particularly suitable in the mentioned method for detecting and/or manipulating a protein of interest, and the application of such particularly labelled substrates. This patent application also describes methods of manufacture of these additional AGT substrates.
Human AGT mutant Gly160Trp (Xu-Welliver et al., Biochemical Pharmacology 58:1279-1285, 1999) is somewhat more reactive towards benzylguanine derivatives than wild type human AGT. Juillerat et al., Chem Biol 10:313-317, 2003, prepared a number of mutants of human AGT in the search for more reactive partners for efficient in vivo (intracellular) labeling of AGT fusion proteins with synthetic substrates. Mutations in position 140, 157, 159 and 160 were reported. The mutant Asn157Gly Ser159Glu shows increased activity against benzylguanine derivatives by a factor of approximately 20 compared to wild type hAGT.
The following additional mutations in hAGT have been shown to disrupt DNA binding of hAGT but do not significantly interfere with the activity against benzylguanine derivatives: Lys125Ala, Ala127Thr and Arg128Ala, see Lim et al., EMBO J 15:4050-4060, 1996, and Daniels et al., EMBO J 19:1719-1730, 2000.
The crystal structure of human AGT (pdb-ID 1EH6; Daniels et al., EMBO J 19:1719, 2000) was resolved using a functional protein that was truncated after Asn207.